The AIDS-associated opportunistic parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. In immunocompromised hosts, such as AIDS patients, this parasite may cause severe, protracted and ultimately fatal disease. There is no specific treatment approved for cryptosporidiosis in AIDS patients and there is no vaccine available to prevent Cryptosporidium infection. Thus, the continued search for novel and effective interventional and preventive modalities is crucial. The overall goal of this project is to investigate the mechanisms by which the innate immune system recognizes and responds to C. parvum. While innate immunity is clearly important, the host cell receptors, parasite ligands and signaling pathways involved in initiating and mediating innate immune responses to C. parvum are unknown. Knowledge about these receptors, ligands and pathways is essential for development of novel and effective preventive and interventional modalities. TLR-mediated pathways (particularly TLRs 2, 4 and 9) have been shown to be important in innate immune responses to several parasites. However, the role of these pathways in innate immunity to C. parvum is unknown. Preliminary studies indicate that these pathways may be involved in resistance to infection with this parasite. The hypothesis is that recognition of C. parvum and consequent initiation of innate immune responses to this parasite is mediated by specific TLRs, in particular TLRs 2, 4 and 9. The specific aim is to determine which TLRs are involved in innate immune responses to C. parvum in human and murine cell lines in vitro and in mice in vivo. In the first part of the specific aim, TLR-mediated pro-inflammatory responses to C. parvum will be investigated in human and murine cell lines in which the expression of individual TLRs and associated co-receptors will be post-transcriptionally silenced using siRNA-based approaches. This strategy will be complemented by assessment of C. parvum induced proinflammatory responses in human cell lines into which specific TLR (and co-receptor) genes have been introduced as well as in cell lines derived from TLR-deficient mice. In the second part of the aim, resistance to C. parvum infection in mice deficient in TLRs 2, 4 and 9 will be determined by comparing fecal oocyst shedding and intestinal parasite burden to that in WT mice. In addition, expression and secretion of proinflammatory cytokines and chemokines will be evaluated in C. parvum-infected and uninfected WT and TLR-deficient mice. This will enable future studies to identify the ligand/s and elucidate the specific signaling pathways involved. The long-term goal is to determine whether specific molecules or pathways can be targeted to enhance innate immunity to the parasite or to prevent immunopathology induced by dysregulated innate immune responses. [unreadable] [unreadable]